Integrin-Arg-SHP2 signaling regulates NMDAR function and neuron morphology

整合素-Arg-SHP2信号调节NMDAR功能和神经元形态

• 批准号: 8781022
• 负责人: Aaron Donald Levy
• 金额: $ 3.63万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781022
• 关键词: Address; Adolescence; Affect; Alleles; Alzheimer' s Disease; Attenuated; Autistic Disorder; Binding; Biochemical; Biotinylation; Cognitive deficits; Confocal Microscopy; Data; Defect; Dendrites; Dendritic Spines; Diagnosis; Disease; Economic Burden; Electron Microscopy; Functional disorder; Genetic; Head; Hippocampus (Brain); Integrins; Knock-in Mouse; Link; Maintenance; Measures; Mediating; Mental Retardation; Mental disorders; Molecular; Mood Disorders; Morphology; Mus; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NMDA receptor antagonist; Neurodegenerative Disorders; Neurons; Non-Receptor Type 11 Protein Tyrosine Phosphatase; Noonan Syndrome; PTPN11 gene; Pathology; Phosphorylation; Phosphorylation Site; Play; Prosencephalon; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Regulation; Regulatory Pathway; Role; Schizophrenia; Signal Transduction; Site; Slice; Structure; Surface; Synapses; Techniques; Testing; Therapeutic Intervention; Tyrosine Phosphorylation Site; Vertebral column; addiction; adhesion receptor; autism spectrum disorder; density; developmental disease; excitatory neuron; gain of function; insight; mutant; nervous system disorder; neuron loss; public health relevance; receptor function; research study; social; treatment strategy

DESCRIPTION (provided by applicant): Integrin-Arg-SHP2 signaling regulates NMDAR function and neuron morphology. Dendritic spine stability is disrupted in psychiatric and neurological disorders. Regulation of NMDA receptor (NMDAR) activity by integrin adhesion receptors plays a fundamental role in dendritic spine maturation and stability, but the molecular mechanisms by which integrins regulate NMDARs are unknown. We have shown that loss of integrin ?3?1 signaling through the Abl2/Arg non-receptor tyrosine kinase causes widespread dendritic spine loss in late adolescence due to increased GluN2B subunit-mediated NMDA receptor currents. I provide strong evidence that Arg acts through the tyrosine phosphatase SHP2 to control GluN2B phosphorylation and function. NMDA receptor dysfunction is a hallmark of psychiatric, neurodevelopmental and neurological diseases such as schizophrenia, autism, and Alzheimer's disease. Understanding the mechanism by which integrins regulate NMDAR function is critical to understand how synaptic stability is compromised in these disorders and to develop treatment strategies. In this proposal, I will test the hypothesis that integrin-Arg-SHP2 signaling functionally regulates the NMDA receptor to control dendritic spine stability. My first aim is to determine how SHP2 regulates GluN2B phosphorylation and function. In this aim, I will use substrate-trapping mutants of SHP2 to test whether GluN2B is directly dephosphorylated by SHP2 and to determine which site in GluN2B is targeted by SHP2. GluN2B phosphorylation promotes its surface localization. Therefore, I will also use surface biotinylation of NMDARs in cultured hippocampal neurons to measure how genetic and pharmacological loss- or gain-of-function of Arg and SHP2 influence NMDAR surface expression. My second aim is to elucidate an integrin-NMDA receptor regulatory mechanism. GluN2B phosphorylation increases NMDAR-mediated currents, and my preliminary data show that activation of SHP2 decreases GluN2B phosphorylation. To test how SHP2 regulates NMDAR function, I will measure how loss- or gain-of-function of SHP2 activity affects NMDAR-mediated currents in hippocampal slices. I will also test the hypothesis that SHP2 functions as a mechanistic link between integrin-Arg signaling and the NMDAR by assessing whether an SHP2 gain-of-function allele can suppress elevated NMDAR currents in mice lacking integrin ?3?1 or Arg. My third aim is to understand how SHP2 regulates dendritic spine and synapse structure. Our lab has shown that integrin-Arg signaling attenuates NMDAR activity to stabilize spines, and I hypothesize that SHP2 mediates the effects of Arg signaling on NMDAR function. I will use confocal and electron microscopy to quantitatively measure how loss- and gain-of-function of SHP2 activity in mice affects dendritic spine and synapse structure and stability. I will also test whether an activated SHP2 allele can suppress the spine destabilization resulting from loss of integrin ?3?1 or Arg in mice.

描述（由申请人提供）：整合素-Arg-SHP2信号传导调节NMDAR功能和神经元形态。精神和神经疾病中树突棘的稳定性受到破坏。整合素粘附受体对 NMDA 受体 (NMDAR) 活性的调节在树突棘成熟和稳定性中起着重要作用，但整合素调节 NMDAR 的分子机制尚不清楚。我们已经证明，由于 GluN2B 亚基介导的 NMDA 受体电流增加，通过 Abl2/Arg 非受体酪氨酸激酶的整合素 ?3?1 信号丢失会导致青春期后期广泛的树突棘丢失。我提供了强有力的证据证明 Arg 通过酪氨酸磷酸酶 SHP2 发挥作用来控制 GluN2B 磷酸化和功能。 NMDA 受体功能障碍是精神分裂症、自闭症和阿尔茨海默病等精神、神经发育和神经系统疾病的标志。了解整合素调节 NMDAR 功能的机制对于了解这些疾病中突触稳定性如何受到损害并制定治疗策略至关重要。在本提案中，我将测试整合素-Arg-SHP2 信号传导功能调节 NMDA 受体以控制树突棘稳定性的假设。我的第一个目标是确定 SHP2 如何调节 GluN2B 磷酸化和功能。为此，我将使用 SHP2 的底物捕获突变体来测试 GluN2B 是否直接被 SHP2 去磷酸化，并确定 GluN2B 中的哪个位点是 SHP2 的目标位点。 GluN2B 磷酸化促进其表面定位。因此，我还将在培养的海马神经元中使用 NMDAR 的表面生物素化来测量 Arg 和 SHP2 的遗传和药理学功能丧失或获得如何影响 NMDAR 表面表达。我的第二个目标是阐明整合素 NMDA 受体的调节机制。 GluN2B 磷酸化会增加 NMDAR 介导的电流，我的初步数据表明 SHP2 的激活会降低 GluN2B 磷酸化。为了测试 SHP2 如何调节 NMDAR 功能，我将测量 SHP2 活性的功能丧失或功能增强如何影响海马切片中 NMDAR 介导的电流。我还将通过评估 SHP2 功能获得等位基因是否可以抑制缺乏整合素 ?3?1 或 Arg 的小鼠中升高的 NMDAR 电流，来检验 SHP2 作为整合素-Arg 信号传导和 NMDAR 之间的机制联系的假设。我的第三个目标是了解 SHP2 如何调节树突棘和突触结构。我们的实验室已经表明，整合素-Arg 信号传导会减弱 NMDAR 活性以稳定脊柱，并且我假设 SHP2 介导 Arg 信号传导对 NMDAR 功能的影响。我将使用共聚焦和电子显微镜来定量测量小鼠中 SHP2 活性的丧失和功能获得如何影响树突棘和突触结构和稳定性。我还将测试激活的 SHP2 等位基因是否可以抑制小鼠中因整合素 ?3?1 或 Arg 丢失而导致的脊柱不稳定。